DESCRIPTION (Investigator's Abstract): Cytochromes P450 play a pivotal role in the detoxification and bioactivation of drugs, environmental contaminants, and numerous other foreign compounds. Under many conditions a single P450 may be exclusively or primarily responsible for the detoxification or bioactivation of a particular chemical. Accordingly, the phenotype of an individual human or experimental animal with respect to the amounts of particular P450 forms expressed in a given organ can determine the metabolism and pharmacological activity or toxicity of a compound. The long-term objective of the proposed research is to elucidate the molecular basis for the substrate specificity of cytochromes P450 2B. These enzymes represent the major phenobarbital-inducible cytochromes P450 in many species, and exhibit some striking differences as well as similarities in substrate specificity. To date, all individual amino acid residues that contribute to P450 2B specificity have been found within five of six proposed substrate recognition sites (SRSs). The hypothesis to be tested is that appropriate combinations of residue substitutions within the SRSs will interconvert the substrate specificities and susceptibility to inhibition of P450 2B enzymes. To address this hypothesis, wild-type and site-directed mutants of rat P450 2B1 and 2B2, rabbit P450 2B4 and 2B5, human P450 2B6, and canine P450 2B11 will be expressed in E. coli and analyzed with a battery of substrates and inhibitors. The results will be interpreted with the help of 3-D homology models based on bacterial cytochromes P450 of known structure. The individual specific aims are: 1) To identify inhibitors that distinguish between P450 2B1 and 2B2 and between P450 2B4 and 2B5 and to determine the amino acid residues responsible for the differences in selectivity. 2) To identify the structural basis for the unique functional properties of human P450 2B6 compared with its counterparts in other species. 3) To determine the mechanistic basis for differences in catalytic activity and inhibitor susceptibility of wild-type and mutant cytochromes P450 2B. The studies should provide a paradigm for predicting and/or rationalizing individual, strain, and species differences in P450 function.